ISKpn28 transposition and genomic alterations in λ-Red recombinants of hypervirulent Klebsiella pneumoniae : Whole-genome sequencing and phenotypic analysis

The λ-Red recombination system is commonly used to investigate gene function in Klebsiella pneumoniae; however, unintended secondary genomic alterations in recombinant strains may remain undetected by locus-specific validation methods such as polymerase chain reaction and Sanger sequencing. Following rmpA mutant construction in the clinical hypervirulent K. pneumoniae (hvKp) strain K255, two recombinant derivatives showed identical results on locus-specific validation, confirming the intended rmpA replacement. However, the strains differed in uronic acid content and interaction with murine macrophages. We therefore performed whole-genome sequencing (WGS) incorporating both short- and long-read data to investigate potential secondary genomic alterations. Both recombinant derivatives harbored an additional chromosomal copy of ISKpn28 within the nlpD gene. The K255ΔrmpA-1 strain additionally exhibited a chromosomal deletion of 55,652 base pairs, whereas K255ΔrmpA-2 harbored an ISKpn74-associated plasmid inversion. These findings provide genomic evidence of ISKpn28 transposition in recombinant derivatives of the clinical hvKp strain K255, and highlight the potential value of WGS incorporating long-read sequencing for assessing genome-wide integrity in λ-Red-derived recombinant hvKp strains harboring ISKpn28.